Abstract The spread of malaria parasites by the Anopheles mosquito vector sustains high levels of reinfection in endemic regions and severely impedes malaria control. As a result, efforts to eliminate malaria must include strategies that prevent parasite transmission. Because only the asexual blood stages of the parasite cause disease, they are the main target of most antimalarials. In contrast, the developmental stage responsible for transmission, the gametocyte, is only poorly understood, and most drugs that are effective against the asexual blood stages are ineffective against gametocytes. Here, we will directly measure and identify the activity of a particularly druggable enzyme family, the serine hydrolases, in the native proteome of Plasmodium falciparum (Pf) schizont and gametocyte stages. We will use activity-based protein profiling (ABPP) with a serine hydrolase-directed activity probe in combination with quantitative mass spectrometry to survey a broad cross- section of enzyme activity during the transition from the asexual to the sexual, mosquito-transmissible form. Because ABPP is a mechanism-based and sequence-independent approach, these studies will also capture the activity of Pf proteins with unknown function. We will further identify the serine proteases among the hits, and define the physiologic substrates of known and previously unknown proteases. Defining the SH activity changes associated with the transition from asexual to sexual Pf gametocytes will provide a new and broad activity-based view of this developmental transition, identify potential transmission blocking targets, and provide experimental annotation for a large number of proteins with unknown function.